The Development Research Of Molecular Diagnostic Technique Based On Nucleic Acid Probe And Its Application In Liquid Biopsy For Prostate Cancer

Prostate cancer is one of the most common malignant tumors in men globally,significantly endangering human health.Traditional diagnostic methods for prostate cancer primarily include prostate-specific antigen(PSA)testing,digital rectal examination,and tissue biopsy.However,these methods exhibit certain limitations,such as low specificity,low sensitivity,and invasiveness.In recent years,DNA methylation testing has emerged as a novel diagnostic approach and has been extensively applied in the field of prostate cancer diagnosis.In tissue samples,DNA methylation testing has demonstrated high diagnostic efficacy and has been recommended by the European Association of Urology(EAU)guidelines.Nevertheless,in body fluid samples such as blood and urine,despite being less invasive,its diagnostic efficacy is relatively inferior.This is primarily due to the extremely low proportion of circulating tumor DNA in plasma,which is easily masked by a large amount of wild-type free DNA.Consequently,the current technology is unable to achieve simple,accurate,rapid,and low-cost detection.Nucleic acid probe technology,characterized by rapidity and high sensitivity,has the potential to overcome these technical bottlenecks.However,challenges still exist in its specific application.This study aims to address the issues of nucleic acid probes in molecular diagnosis,focusing on DNA methylation mutations as monitoring targets,and to explore an accurate,rapid,and low-cost liquid biopsy method for prostate cancer diagnosis.The research initially addressed the bottleneck issues in the two major types of nucleic acid probe systems used in the Post-PCR phase: enzyme-assisted signal amplification systems and branch migration probe systems.In the enzyme-assisted signal amplification system,a novel guide-strand-assisted mutation detection system was developed,significantly reducing side activities to enhance specificity while maintaining the main activity to ensure sensitivity.In the branch migration system,a novel leakage control strategy based on a DNA triplex structure was developed to effectively inhibit leakage reactions.Subsequently,these two nucleic acid probes were applied to DNA methylation detection,and hold strands were introduced to optimize the methylation-specific PCR enrichment process,constructing a DNA methylation detection platform with a detection limit as low as 0.002%.Finally,the DNA methylation detection platform was applied to prostate cancer liquid biopsy,achieving a detection sensitivity of 89% and specificity of 76%.